Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia.
<h4>Background</h4>Ethiopia is one of the high multidrug-resistant tuberculosis (MDR-TB) burden countries. However, phenotypic drug susceptibility testing can take several weeks due to the slow growth of Mycobacterium tuberculosis complex (MTBC) strains. In this study, we assessed the pe...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2022-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0271508 |
| _version_ | 1828452603100397568 |
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| author | Eyob Abera Mesfin Matthias Merker Dereje Beyene Abreham Tesfaye Yassir Adam Shuaib Desalegn Addise Belay Tessema Stefan Niemann |
| author_facet | Eyob Abera Mesfin Matthias Merker Dereje Beyene Abreham Tesfaye Yassir Adam Shuaib Desalegn Addise Belay Tessema Stefan Niemann |
| author_sort | Eyob Abera Mesfin |
| collection | DOAJ |
| description | <h4>Background</h4>Ethiopia is one of the high multidrug-resistant tuberculosis (MDR-TB) burden countries. However, phenotypic drug susceptibility testing can take several weeks due to the slow growth of Mycobacterium tuberculosis complex (MTBC) strains. In this study, we assessed the performance of a Sanger sequencing approach to predict resistance against five anti-tuberculosis drugs and the pattern of resistance mediating mutations.<h4>Methods</h4>We enrolled 226 MTBC culture-positive MDR-TB suspects and collected sputum specimens and socio-demographic and TB related data from each suspect between June 2015 and December 2016 in Addis Ababa, Ethiopia. Phenotypic drug susceptibility testing (pDST) for rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin using BACTEC MGIT 960 was compared with the results of a Sanger sequencing analysis of seven resistance determining regions in the genes rpoB, katG, fabG-inhA, pncA, embB, rpsL, and rrs.<h4>Result</h4>DNA isolation for Sanger sequencing was successfully extracted from 92.5% (209/226) of the MTBC positive cultures, and the remaining 7.5% (17/226) strains were excluded from the final analysis. Based on pDST results, drug resistance proportions were as follows: isoniazid: 109/209 (52.2%), streptomycin: 93/209 (44.5%), rifampicin: 88/209 (42.1%), ethambutol: 74/209 (35.4%), and pyrazinamide: 69/209 (33.0%). Resistance against isoniazid was mainly mediated by the mutation katG S315T (97/209, 46.4%) and resistance against rifampicin by rpoB S531L (58/209, 27.8%). The dominating resistance-conferring mutations for ethambutol, streptomycin, and pyrazinamide affected codon 306 in embB (48/209, 21.1%), codon 88 in rpsL (43/209, 20.6%), and codon 65 in pncA (19/209, 9.1%), respectively. We observed a high agreement between phenotypic and genotypic DST, such as 89.9% (at 95% confidence interval [CI], 84.2%-95.8%) for isoniazid, 95.5% (95% CI, 91.2%-99.8%) for rifampicin, 98.6% (95% CI, 95.9-100%) for ethambutol, 91.3% (95% CI, 84.6-98.1%) for pyrazinamide and 57.0% (95% CI, 46.9%-67.1%) for streptomycin.<h4>Conclusion</h4>We detected canonical mutations implicated in resistance to rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin. High agreement with phenotypic DST results for all drugs renders Sanger sequencing promising to be performed as a complementary measure to routine phenotypic DST in Ethiopia. Sanger sequencing directly from sputum may accelerate accurate clinical decision-making in the future. |
| first_indexed | 2024-12-10T23:54:02Z |
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| language | English |
| last_indexed | 2024-12-10T23:54:02Z |
| publishDate | 2022-01-01 |
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| spelling | doaj.art-79bd28c709a94d46838c7da188ed38672022-12-22T01:28:38ZengPublic Library of Science (PLoS)PLoS ONE1932-62032022-01-01178e027150810.1371/journal.pone.0271508Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia.Eyob Abera MesfinMatthias MerkerDereje BeyeneAbreham TesfayeYassir Adam ShuaibDesalegn AddiseBelay TessemaStefan Niemann<h4>Background</h4>Ethiopia is one of the high multidrug-resistant tuberculosis (MDR-TB) burden countries. However, phenotypic drug susceptibility testing can take several weeks due to the slow growth of Mycobacterium tuberculosis complex (MTBC) strains. In this study, we assessed the performance of a Sanger sequencing approach to predict resistance against five anti-tuberculosis drugs and the pattern of resistance mediating mutations.<h4>Methods</h4>We enrolled 226 MTBC culture-positive MDR-TB suspects and collected sputum specimens and socio-demographic and TB related data from each suspect between June 2015 and December 2016 in Addis Ababa, Ethiopia. Phenotypic drug susceptibility testing (pDST) for rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin using BACTEC MGIT 960 was compared with the results of a Sanger sequencing analysis of seven resistance determining regions in the genes rpoB, katG, fabG-inhA, pncA, embB, rpsL, and rrs.<h4>Result</h4>DNA isolation for Sanger sequencing was successfully extracted from 92.5% (209/226) of the MTBC positive cultures, and the remaining 7.5% (17/226) strains were excluded from the final analysis. Based on pDST results, drug resistance proportions were as follows: isoniazid: 109/209 (52.2%), streptomycin: 93/209 (44.5%), rifampicin: 88/209 (42.1%), ethambutol: 74/209 (35.4%), and pyrazinamide: 69/209 (33.0%). Resistance against isoniazid was mainly mediated by the mutation katG S315T (97/209, 46.4%) and resistance against rifampicin by rpoB S531L (58/209, 27.8%). The dominating resistance-conferring mutations for ethambutol, streptomycin, and pyrazinamide affected codon 306 in embB (48/209, 21.1%), codon 88 in rpsL (43/209, 20.6%), and codon 65 in pncA (19/209, 9.1%), respectively. We observed a high agreement between phenotypic and genotypic DST, such as 89.9% (at 95% confidence interval [CI], 84.2%-95.8%) for isoniazid, 95.5% (95% CI, 91.2%-99.8%) for rifampicin, 98.6% (95% CI, 95.9-100%) for ethambutol, 91.3% (95% CI, 84.6-98.1%) for pyrazinamide and 57.0% (95% CI, 46.9%-67.1%) for streptomycin.<h4>Conclusion</h4>We detected canonical mutations implicated in resistance to rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin. High agreement with phenotypic DST results for all drugs renders Sanger sequencing promising to be performed as a complementary measure to routine phenotypic DST in Ethiopia. Sanger sequencing directly from sputum may accelerate accurate clinical decision-making in the future.https://doi.org/10.1371/journal.pone.0271508 |
| spellingShingle | Eyob Abera Mesfin Matthias Merker Dereje Beyene Abreham Tesfaye Yassir Adam Shuaib Desalegn Addise Belay Tessema Stefan Niemann Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia. PLoS ONE |
| title | Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia. |
| title_full | Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia. |
| title_fullStr | Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia. |
| title_full_unstemmed | Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia. |
| title_short | Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia. |
| title_sort | prediction of drug resistance by sanger sequencing of mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in ethiopia |
| url | https://doi.org/10.1371/journal.pone.0271508 |
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